The compound of the formula: ##STR1## wherein R1 is hydrogen, methyl or hydroxymethyl, R2 is hydrogen or methoxy, R3 is hydroxy or methoxy, and R4 is hydrogen or a group ##STR2## is novel, and the compound is of use as inhibitors of animal tissue fibrosis, or biochemical reagents.
|
1. A compound represented by the formula: ##STR16## wherein R1 is hydrogen, methyl or hydroxymethyl, R2 is hydrogen or methoxy, R3 is hydroxy or methoxy and R4 is hydrogen or a group ##STR17##
2. A compound as claimed in
3. A compound as claimed in
4. A compound as claimed in
5. A compound as claimed in
6. A compound as claimed in
7. A compound as claimed in
8. A compound as claimed in
9. A compound as claimed in
10. A compound as claimed in
11. A compound as claimed in
12. A compound as claimed in
13. A compound as claimed in
14. An antifibrotic preparation comprising an effective amount of at least one compound of
pharmaceutically acceptable carrier, excipient or diluent. 15. A compound represented by the formula: ##STR25## wherein R1 is hydrogen, methyl or hydroxymethyl, R2 is hydrogen or methoxy, R3 is hydroxy or methoxy and R4 is a group ##STR26## 16. A compound as claimed in claim 15, wherein R1 is methyl, R2 is hydrogen and R3 is methoxy. 17. A compound as claimed in claim 15, wherein R1 is methyl, R2 is methoxy and R3 is methoxy. 18. A compound as claimed in claim 15, wherein R1 is hydrogen, R2 is methoxy and R3 is methoxy. 19. A compound as claimed in claim 15, wherein R1 is methyl, R2 is methoxy and R3 is hydroxy. 20. A compound as claimed in claim 15, wherein R1 is hydroxymethyl, R2 is hydrogen and R3 is methoxy. 21. A compound as claimed in claim 15, wherein R1 is hydroxymethyl, R2 is methoxy and R3 is methoxy. 22. A compound represented by the formula: ##STR27## 23. A compound represented by the formula: ##STR28## 24. A compound represented by the formula: ##STR29## 25. A compound represented by the formula: ##STR30## 26. An antifibrotic preparation comprising an effective amount of at least one compound of any one of claims 15 or 22-25, and a pharmaceutically acceptable carrier, excipient or diluent. |
|||||||||||||||||||||||||||||||||||
The present invention relates to Physiologically Active Substance P-23924, its production and use.
Protocollagen prolyl hydroxylase is an enzyme which specifically hydroxylates the proline moiety of the protocollagen synthesized by ribosome in animal cells and is an important and rate-limiting factor in the biosynthesis of collagen. Among the compounds known to inhibit their enzyme activity are iron chelators (e.g. α,α'-dipyridyl etc.). SH enzyme inhibitors (e.g. p-chloromercury benzoate etc.) and certain heavy metals (e.g. Cu++, Zn++, etc.). However, because these substances invariably inhibit collagen biosynthesis non-specifically, they cause serious side effects and cannot be used as drugs. If a substance was found that would not inhibit biosynthesis of non-collagenous proteins but would specifically inhibit collagen biosynthesis, such substance could be used for the prevention and treatment of organ fibrosis accompanied by an excessive accumulation of collagen such as arteriosclerosis, liver cirrhosis, scleroderma, keloid, rheumatic arthritis, pulmonary fibrosis, etc.
In their search for a substance capable of inhibiting protocollagen prolyl hydroxylase activity among microbial metabolites, the present inventors discovered that an actinomycete strain produces Physiologically Active Substance P-23924A, B, C, D, E and F which are novel inhibitors of collagen biosynthesis and that these compounds are convertible to their reduced forms. This finding was followed by further research which has resulted in the perfection of the present invention.
The present invention relates to (1) a compound represented by the formula: ##STR3## wherein R1 is hydrogen, methyl or hydroxymethyl, R2 is hydrogen or methoxy, R3 is hydroxy or methoxy and R4 is hydrogen or a group ##STR4##
(2) a method of producing Physiologically Active Substance P-23924 A, B, C, D, E and/or F which comprises cultivating a microorganism belonging to the genus Streptomyces and capable of elaborating Physiologically Active Substance P-23924 A, B, C, D, E and/or F in a medium to cause said microorganism to elaborate and accumulate Physiologically Active Substance P-234924 A, B, C, D, E and/or F and recovering the same substance from the resulting culture broth.
(3) a method of producing a compound represented by the formula ##STR5## wherein R1 is hydrogen, methyl or hydroxymethyl, R2 is hydrogen or methoxy, and R3 is hydroxy or methoxy, which comprises subjecting to reduction a compound of the formula: ##STR6## wherein R1, R2 and R3 have the same meanings ad defined above, and
(4) an antifibriotic preparation containing Physiologically Active Substance P-23924.
The compounds of the compound (I) are named as follows:
| __________________________________________________________________________ |
| Name Abbreviation |
| R1 |
| R2 |
| R3 |
| R4 |
| __________________________________________________________________________ |
| Physiologically Active Substance P-23924A |
| P-23924A |
| CH3 |
| H OCH3 |
| ##STR7## |
| Physiologically Active Substance P-23924B |
| P-23924B |
| CH3 |
| OCH3 |
| OCH3 |
| ##STR8## |
| Physiologically Active Substance P-23924C |
| P-23924C |
| H OCH3 |
| OCH3 |
| ##STR9## |
| Physiologically Active Substance P-23924D |
| P-23924D |
| CH3 |
| OCH3 |
| OH |
| ##STR10## |
| Physiologically Active Substance P-23924E |
| P-23924E |
| CH2 OH |
| H OCH3 |
| ##STR11## |
| Physiologically Active Substance P-23924F |
| P-23924F |
| CH2 OH |
| OCH3 |
| OCH3 |
| ##STR12## |
| Physiologically |
| P-23924AR |
| CH3 |
| H OCH3 |
| H |
| Active Substance |
| P-23924AR |
| Physiologically |
| P-23924BR |
| CH3 |
| OCH3 |
| OCH3 |
| H |
| Active Substance |
| P-23924BR |
| Physiologically |
| P-23924CR |
| H OCH3 |
| OCH3 |
| H |
| Active Substance |
| P-23924CR |
| Physiologically |
| P-23924DR |
| CH3 |
| OCH3 |
| OH H |
| Active Substance |
| P-23924DR |
| __________________________________________________________________________ |
Moreover, the compounds of the formula (I) will sometimes be referred to generically or individually as Physiologically Active Substances P-23924 or briefly as P-23924.
The microorganism employed in accordance with the present invention may be any strain of microorganism that belongs to the genus Streptomyces and is able to elaborate Physilogically Active Substance P-23924 A, B, C, D, E and/or F (which will sometimes be referred to briefly as a P-23924 producing strain.
As an Example of the P-23924 producing strain, there may be mentioned Streptomyces sp. No. 23924 (hereinafter referred to briefly as Strain No. 23924) which was isolated from a soil sample collected in Ishigakijima, Okinawa Prefecture, Japan.
The characteristics of this Strain No. 23924 as determined in accordance with the methods described in International Journal of Systematic Bacteriology 16, No. 3, pp. 313-340 (1966) are as follows. Unless otherwise indicated, cultural characteristics were those found after 14 days incubation at 28°C
Monopodially extending from vegetative mycelia are short sporulated filaments which are spiral or sometimes incompletely spiral, open-loop or hook-shaped, without whorl formation. Mature spore chains generally show 5 to 10 spores in chains. The spores are either cylindrical or elipsoidal and range in size from 0.4 to 0.7 by 0.7 to 1.2 microns, with smooth surfaces.
The degree of growth (G), growth and color of aerial mycelium (AM) and the production and color of soluble pigments (SP), among others, on various media are listed below. As to descriptions of colors, the standard color codes given in the Color Harmony Manual, Fourth Edition (1958) from Container Corporation of America were employed.
| ______________________________________ |
| (a) Sucrose nitrate agar |
| (G): Poor |
| (AM): Sparse, light grayish brown (3 ge) |
| (SP): None |
| (b) Glucose asparagine agar |
| (G): Poor |
| (AM): None |
| (SP): None |
| (c) Glycerin asparagine agar |
| (G): None |
| (d) Starch inorganic salt agar |
| (G): Good |
| (AM): Abundant, powdery, (3 ih) |
| (SP): Yellowish brown (4 gc) |
| (e) Tyrosine agar |
| (G): Poor |
| (AM): Sparse, gray (3 ba) |
| (SP): Light yellowish red (4 ea) |
| (f) Nutrient agar |
| (G): Moderate |
| (AM): None |
| (SP): None |
| (g) Yeast malt agar |
| (G): Moderate |
| (AM): Sparse, gray (3 fe) |
| (SP): Light yellowish brown (4 ne) |
| (h) Oatmeal agar |
| (G): Moderate |
| (AM): Sparse, light grayish brown (3 ge) |
| (SP): None |
| (III) Physiological characteristics |
| (a) Temperature range for growth: 15-35°C |
| (b) Liquefaction of gelatin (glucose peptone gelatin, |
| 24°C, 3 weeks): positive (weak) |
| (c) Hydrolysis of starch: positive |
| (d) Coagulation and peptonization of skim milk: |
| both negative |
| (e) Reduction of nitrate: |
| negative |
| (f) Production of melanoid pigment: |
| Tyrosine agar: suspected positive |
| Peptone yeast iron agar: negative |
| (IV) Assimilation of carbon sources (Pridham-Gottlieb agar) |
| L-arabinose ± Inositol - |
| D-xylose ++ L-rhamnose |
| - |
| D-glucose ++ Raffinose - |
| D-fructose ± D-mannit - |
| Sucrose ± Control - |
| ______________________________________ |
| (Note) |
| ++: Good growth |
| ±: Slight growth |
| -: No growth |
It is apparent from the above descriptions of characteristics that this particular strain belongs to the genus Streptomyces.
The above Streptomyces sp. No. 23924 strain has been deposited at Institute for Fermentation, Osaka (IFO), Japan under the accession number of IFO 14205 since Sept. 20, 1982, and this microorganism, which was deposited on Oct. 1, 1982 at Fermentation Research Institute Agency of Industrial science and Technology, Ministry of International Trade and Industry (FRI), Japan under the accession number of FERM P-6739, the deposit being converted to a deposit under the Budapest Treaty, has been stored at FRI under the accession number of FERM BP-338.
Microorganisms of the genus Streptomyces are generally liable to change in characteristics and can be easily caused to undergo mutation by artificial procedures such as X-ray, ultraviolet ray or radioactive ray irradiation or by a treatment employing a mutagenic agent. Such mutants, only if they are able to produce P-23924 A, B, C, D, E and/or F, can all be employed for the purposes of the present invention.
The medium for cultivation of P-23924 producing strains may be a liquid medium or a solid medium and it is generally advantageous to conduct shake culture or aerobic submerged culture using a liquid medium. The medium may be of any kind only if it is suitable for growth of Actinomyces and for the production thereby of P-23924A, B, C, D, E and/or F. Thus, as sources of carbon, there may be employed glucose, lactose, glycerin, starch, sucrose, dextrin, molasses, organic acids (e.g. acetic acid, tartaric acid, etc.) and so on. As sources of nitrogen, there may be employed peptone, Casamino acid (Difco, U.S.A.), N-Z Amine A (Sheffield, U.S.A.) and other protein hydrolysates, yeast extract, malt extract, soybean meal, corn steep liquor, amino acids (e.g. aspartic acid, glutamic acid, etc.), various ammonium salts (e.g. ammonium sulfate, ammonium chloride, etc.) and so on. It is also possible to add inorganic salts such as various phosphates (e.g. sodium dihydrogen phosphate, potassium monohydrogen phosphate, etc.), metal salts (e.g. magnesium sulfate, sodium chloride, ferrous sulfate, etc.), heavy metal salts (e.g. manganese sulfate, zinc sulfate, etc.) and so on. For the purpose of promoting growth of the strain, vitamins (e.g. vitamin B1, calcium pantothenate, etc.) nucleic acid related products (e.g. adenine, uracil, etc.), etc. may be added. Moreover, according to the cultural method and conditions used, the output of P-23924A, B, C, D, E and/or F may at times be increased by adding a defoaming agent such as silicone, polypropylene glycol ether derivative [e.g. Actocol (Takeda Chemical Industries, Ltd., Japan), etc.], soybean oil, etc. to the culture medium.
When the cultivation temperature and time, the pH of the medium, and other cultural conditions depend on the particular strain and the composition of the medium, they should be so selected and controlled that the output of P-23924 A, B, C, D, E and/or F will be maximal. It is in many cases desirable to conduct aerobic culture at about 20° to 40°C for 24 to 240 hours while the pH of the medium is maintained at about 4 to 9. Recovery of Physiologically Active Substance P-23924 A, B, C, D, F and/or F from the culture broth can be easily accomplished by utilizing various procedures in a combination suited to the properties of the same substance. Among such procedure are extraction with an organic solvent which is neutral or weakly acidic and immiscible with water, such as ethyl acetate, butyl acetate, chloroform, butanol, benzene, toluene, diethyl ether, methylene chloride, methyl isobutyl ketone, etc., adsorption chromatography using activated carbon, silica gel, alumina, etc., gel filtration on a Sephadex column, ion exchange chromatography using ion exchange resins, and so on. By applying such procedures in a suitable combination, P-23924 A, B, C, D, E and/or F can be isolated as crystals or crystalline powders.
Various properties of Physiologically Active Substance P-23924 A, B, C, D, E and/or F as obtained in Example 2 which appears hereinafter are as follows.
(1) Physicochemical properties
(a) Melting point: A 186° to 188°C; B 200° to 202°C; C 187° to 190°C; D 205° to 207°C; E 174° to 176°C; F 191° to 195°C
(b) Elemental analysis (Found):
| ______________________________________ |
| P-23924 |
| C H N S |
| ______________________________________ |
| A 54.75 ± 1.0 |
| 4.92 ± 0.5 |
| 3.77 ± 0.5 |
| 8.09 ± 0.5 |
| B 53.74 ± 1.0 |
| 4.99 ± 0.5 |
| 3.26 ± 0.5 |
| 7.29 ± 0.5 |
| C 52.43 ± 1.0 |
| 4.72 ± 0.5 |
| 3.50 ± 0.5 |
| 7.82 ± 0.5 |
| D 52.58 ± 1.0 |
| 4.61 ± 0.5 |
| 3.55 ± 0.5 |
| 7.84 ± 0.5 |
| E 52.18 ± 1.0 |
| 4.58 ± 0.5 |
| 3.19 ± 0.5 |
| 7.55 ± 0.5 |
| F 51.67 ± 1.0 |
| 4.75 ± 0.5 |
| 3.45 ± 0.5 |
| 7.30 ± 0.5 |
| ______________________________________ |
(c) Molecular weight (based on mass spectrum) and molecular formula:
| ______________________________________ |
| P-23924 Mol. wt. Mol. formula |
| ______________________________________ |
| A 393 C18 H19 NO7 S |
| B 423 C19 H21 NO8 S |
| C 409 C18 H19 NO8 S |
| D 409 C18 H19 NO8 S |
| E 409 C18 H19 NO8 S |
| F 439 C19 H21 NO9 S |
| ______________________________________ |
(d) Specific rotation:
| ______________________________________ |
| P-23924 [α]D 23 (in methanol) |
| C (%) |
| ______________________________________ |
| A -91 ± 10° |
| 0.50 |
| B -90 ± 10° |
| 0.51 |
| C -93 ± 10° |
| 0.51 |
| D -62 ± 10° |
| 0.51 |
| E -86 ± 10° |
| 0.50 |
| F -91 ± 10° |
| 0.51 |
| ______________________________________ |
(e) Solubility:
P-23924A, B, D and F are easily soluble in dimethyl sulfoxide, dimethylformamide, pyridine and 5% aqueous sodium hydrogen carbonate; soluble in methanol, dioxane and acetic acid; slightly soluble or insoluble in water, acetone, chloroform, n-hexane and petroleum ether. P-23924C is easily soluble in dimethyl sulfoxide, dimethylformamide, pyridine and 5% aqueous sodium hydrogen carbonate, soluble in methanol, dioxane, acetone and acetic acid; and slightly soluble or insoluble in water, chloroform, n-hexane and petroleum ether.
P-23924E is easily soluble in dimethyl sulfoxide, dimethylformamide, pyridine, 5% aqueous sodium hydrogen carbonate and methanol, soluble in dioxane and acetic acid, and slightly soluble or insoluble in water, chloroform; n-hexane and petroleum ether.
(f) Ultraviolet and visible absorption spectra:
See FIGS. 1 through 6. The wavelengths (nm) and E1 cm1% values giving absorption peaks immediately after dissolution in the respective solvents are presented in Table 1. In FIGS. 1 through 6,------represents the values measured in methanol,------the values in 0.1N HCl-90% aqueous methanol,------the values measured in 0.1N NaOH-90% aqueous methanol.
| TABLE 1 |
| __________________________________________________________________________ |
| P-23924 |
| Solvent |
| Wave length |
| (Elcm 1%) |
| Wave length |
| (Elcm 1%) |
| Wave length |
| (Elcm 1%) |
| __________________________________________________________________________ |
| A B C |
| Methanol |
| 220 ± 2 |
| (886 ± 90) |
| 220 ± 2 |
| (793 ± 80) |
| 221 ± 2 |
| (877 ± 90) |
| 263 ± 2(sh) |
| (421 ± 45) |
| 262 ± 2(sh) |
| (392 ± 40) |
| 260 ± 2(sh) |
| (398 ± 40) |
| 270 ± 2 |
| (458 ± 50) |
| 268 ± 2 |
| (428 ± 40) |
| 265 ± 2 |
| (415 ± 40) |
| 420 ± 10 |
| (129 ± 10) |
| 308 ± 2 |
| (214 ± 20) |
| 306 ± 2 |
| (256 ± 25) |
| 420 ± 10 |
| 420 ± 10 |
| (108 ± 10) |
| (124 ± 10) |
| 0.1N HCl- |
| 221 ± 2 |
| (914 ± 90) |
| 220 ± 2 |
| (789 ± 80) |
| 221 ± 2 |
| (866 ± 90) |
| 90% aq. |
| 264 ± 2(sh) |
| (442 ± 45) |
| 261 ± 2(sh) |
| (390 ± 40) |
| 260 ± 2(sh) |
| (403 ± 40) |
| methanol |
| 270 ± 2 |
| (476 ± 50) |
| 268 ± 2 |
| (431 ± 45) |
| 266 ± 2 |
| (414 ± 40) |
| 420 ± 10 |
| (133 ± 15) |
| 308 ± 2 |
| (210 ± 20) |
| 306 ± 2 |
| (250 ± 25) |
| 420 ± 10 |
| (107 ± 10) |
| 420 ± 10 |
| (119 ± 10) |
| 0.1N NaOH- |
| 216 ± 2 |
| (505 ± 50) |
| 216 ± 2 |
| (482 ± 50) |
| 216 ± 2 |
| (500 ± 50) |
| 90% aq. |
| 238 ± 2 |
| (681 ± 70) |
| 237 ± 2 |
| (748 ± 75) |
| 236 ± 2 |
| (791 ± 80) |
| methanol |
| 277 ± 2 |
| (248 ± 25) |
| 284 ± 2 |
| (215 ± 20) |
| 286 ± 2 |
| (213 ± 20) |
| 540 ± 10 |
| (131 ± 15) |
| 540 ± 10 |
| (128 ± 15) |
| 540 ± 10 |
| (142 ± 15) |
| See FIG. 1 See FIG. 2 See FIG. 3 |
| D E F |
| Methanol |
| 220 ± 2 |
| (764 ± 80) |
| 221 ± 2 |
| (851 ± 90) |
| 222 ± 2 |
| (866 ± 90) |
| 271 ± 2 |
| (450 ± 45) |
| 263 ± 2(sh) |
| (393 ± 40) |
| 262 ± 2(sh) |
| (379 ± 40) |
| 306 ± 2 |
| (209 ± 20) |
| 271 ± 2 |
| (435 ± 45) |
| 268 ± 2 |
| (381 ± 40) |
| 420 ± 10 |
| (102 ± 10) |
| 420 ± 10 |
| (128 ± 10) |
| 308 ± 2 |
| (205 ± 20) |
| 420 ± 15 |
| (91 ± 10) |
| 0.1N NCl- |
| 220 ± 2 |
| (762 ± 80) |
| 221 ± 2 |
| (869 ± 90) |
| 221 ± 2 |
| (852 ± 90) |
| 90% aq. |
| 271 ± 2 |
| (456 ± 45) |
| 264 ± 2(sh) |
| (411 ± 40) |
| 262 ± 2(sh) |
| (374 ± 40) |
| methanol |
| 306 ± 2 |
| (207 ± 20) |
| 271 ± 2 |
| (445 ± 50) |
| 267 ± 2 |
| (383 ± 40) |
| 420 ± 10 |
| (99 ± 10) |
| 420 ± 10 |
| (127 ± 15) |
| 307 ± 2 |
| (200 ± 20) |
| 415 ± 15 |
| (107 ± 10) |
| 0.1N NaOH- |
| 216 ± 2 |
| (438 ± 45) |
| 216 ± 2 |
| (478 ± 50) |
| 222 ± 2(sh) |
| (548 ± 60) |
| 90% aq. |
| 234 ± 2 |
| (576 ± 60) |
| 238 ± 2 |
| (633 ± 65) |
| 236 ± 2 |
| (730 ± 70) |
| methanol |
| 299 ± 2 |
| (531 ± 55) |
| 287 ± 2 |
| (231 ± 25) |
| 270 ± 2 |
| (257 ± 30) |
| 540 ± 20 |
| (89 ± 10) |
| 540 ± 10 |
| (122 ± 15) |
| 542 ± 20 |
| (115 ± 10) |
| See FIG. 4 See FIG. 5 See FIG. 6 |
| __________________________________________________________________________ |
| Sh = shoulder |
| ______________________________________ |
| (i) P-23924A |
| 3300, 2930, 1710, 1665, 1640, |
| 1620, 1570, 1540, 1500, 1460, |
| 1420, 1380, 1335, 1280, 1240, |
| 1200, 1180, 1110, 1040, 1000, |
| 970, 900, 860, 800 |
| See FIG. 7. |
| (ii) P-23924B |
| 3300, 2950, 1725, 1705, 1660, |
| 1630, 1590, 1540, 1460, 1420, |
| 1370, 1330, 1300, 1270, 1210, |
| 1190, 1170, 1130, 1100, 1060, |
| 1020, 980, 950, 890, 860, |
| 790, 760, 700 |
| See FIG. 8. |
| (iii) P-23924C |
| 3400, 3070, 2940, 1720, 1680, |
| 1635, 1600, 1540, 1490, 1460, |
| 1420, 1380, 1350, 1310, 1250, |
| 1220, 1180, 1120, 1100, 1040, |
| 990, 930, 880, 820, 800, |
| 705 |
| See FIG. 9. |
| (iv) P-23924D |
| 3400, 3300, 2960, 1720, 1670, |
| 1635, 1610, 1540, 1440, 1380, |
| 1340, 1300, 1260, 1220, 1205, |
| 1130, 1100, 1060, 1000, 900, |
| 870, 820, 800, 780, 690 |
| See FIG. 10. |
| (v) P-23924E |
| 3550, 3300, 3080, 2950, 1725, |
| 1660, 1640, 1610, 1570, 1540, |
| 1490, 1455, 1420, 1380, 1325, |
| 1300, 1240, 1210, 1180, 1120, |
| 1080, 1040, 1010, 940, 920, |
| 860, 820, 800, 770, 700 |
| See FIG. 11. |
| (vi) P-23924F |
| 3300, 1720, 1680, 1660, 1630, |
| 1600, 1530, 1490, 1460, 1420, |
| 1380, 1335, 1310, 1260, 1220, |
| 1190, 1130, 1100, 1030, 1000, |
| 980, 955, 870, 700 |
| See FIG. 12. |
| ______________________________________ |
(h) Color reactions:
All species of P-23924 A, B, C, D, E and F give positive ferric chloride, Rydon-Smith, alcoholic magnesium acetate reactions, and negative ninhydrin and Ehrlich reactions.
(i) Description:
All species of P-23924 A, B, C, D, E and F are acidic and fat-soluble substances occurring as crystals, crystaline powders or powders which are yellow to yellow-orange or orange-yellow in color.
(j) Nuclear magnetic resonance spectra:
The NMR spectra determined in dimethyl-d6 sulfoxide at 400 MHz are shown in FIGS. 13 through 18. Characteristics signals are given below.
(i) P-23924A
δDMSO-d6 ppm: 1.86(3H, s), 2.09(3H, d, J=1.6 Hz). 2.71(1H, dd, J=8.8, 13.6 Hz), 2.91(1H, dd, J=5.0, 13.6 Hz), 3.66(1H, d, J=12.9 Hz), 3.78(1H, d, J=12.9 Hz), 3.96(3H, s), 4.47(1H, dt like, J=5.0, 8.3, 8.8 Hz), 6.89(1H, q, J=1.6 Hz), 7.09(1H, s), 8.18(1H, d, J=8.3 Hz), 12.40(1H, s) See FIG. 13.
(ii) P-23924B
δDMSO-d6 ppm: 1.86(3H, s), 1.95(3H, s), 2.70(1H, dd, J=8.8, 13.7 Hz), 2.89(1H, dd, J=4.9, 13.7 Hz), 3.65(1H, d, J=13.1 Hz), 3.77(1H, d, J=13.1 Hz), 3.95(3H, s), 4.03(3H, s), 4.47(1H, dt like, J=4.9, 8.1, 8.8 Hz), 7.11(1H, s), 8.18(1H, d, J=8.1 Hz), 12.59(1H, s) See FIG. 14.
(iii) P-23924C
δDMSO-d6 ppm: 1.85(3H, s), 2.71(1H, dd, J=8.8, 13.7 Hz), 2.91(1H, dd, J=4.9, 13.7 Hz), 3.66 (1H, d, J=12.9 Hz), 3.77(1H, d, J=12.9 Hz), 3.88 (3H, s), 3.95(3H, s), 4.47(1H, dt like, J=4.9, 8.1, 8.8 Hz), 6.26(1H, s), 7.15(1H, s), 8.18(1H, d, J=8.1 Hz), 12.73(1H, s) See FIG. 15.
(iv) P-23924D
δDMSO-d6 ppm: 1.85(3H, s), 1.94(3H, s), 2.73(1H, dd, J=8.8, 13.5 Hz), 2.92(1H, dd, J=4.8, 13.5 Hz), 3.64(1H, d, J=12.8 Hz), 3.76(1H, d, J=12.8 Hz), 4.00(3H, s), 4.48(1H, dt like, J=4.8, 8.1, 8.8 Hz), 7.03(1H, s), 8.17(1H, d, J=8.1 Hz), 12.79 (1H, s) See FIG. 16.
(v) P-23924E
δDMSO-d6 ppm: 1.85(3H, s), 2.71(1H, dd, J=8.5, 13.7 Hz), 2.90(1H, dd, J=4.9, 13.7 Hz), 3.67 (1H, d, J=13.1 Hz), 3.79(1H, d, J=13.1 Hz), 3.98 (3H, s), 4.47(2H, d, J=2.2 Hz), 4.47(1H, dt like, J=4.9, 8.1, 8.5 Hz), 5.47(1H, s), 6.82(1H, t, J=2.2 Hz), 7.15(1H, s), 8.18(1H, d, J=8.1 Hz), 12.29 (1H, s) See FIG. 17.
(vi) P-23924F
δDMSO-d6 ppm: 1.85(3H, s), 2.70(1H, dd, J=8.8, 13.7 Hz), 2.9(1H, dd, J=4.9, 13.7 Hz), 3.67(1H, H, d, J=13.1Hz), 3.80(1H, d, J=13.1 Hz), 3.97(3H, s), 4.10 (3H, s), 4.36(2H, d, J=3.7 Hz), 4.47(1H, dt like, J=4.9, 8.3, 8.8 Hz), 4.93(1H, br t), 7.16(1H, s), 8.20(1H, d, J=8.3 Hz), 12.70(1H, s) See FIG. 18.
(k) Rf values on thin layer chromatograms:
The Rf values on silica gel plates (Merck, Article No. 5729, West Germany) and reverse-phase HPTLC plates (RP-8, Merck, Article No. 13725, West Germany) are as follows.
| ______________________________________ |
| P-23924 |
| Solvent A B C D E F |
| ______________________________________ |
| (1) Silica gel plate, |
| 0.39 0.42 0.30 0.19 0.12 0.22 |
| chloroform-acetic |
| acid (8:2) |
| i-Propanol-acetic |
| 0.45 0.48 0.29 0.57 0.45 0.39 |
| acid (96:4) |
| (2) Reverse-phase |
| 0.61 0.55 0.69 0.60 0.75 0.72 |
| plate, methanol- |
| water (7:3) |
| ______________________________________ |
Since there is not known any other compound having the above properties, P-23924 A, B, C, D, E and F are considered to be a novel substances.
Based on the above physicochemical properties, the following structural formula is proposed for P-23924 A, B, C, D, E and F.
| ______________________________________ |
| ##STR13## |
| Compound R1 R2 R3 |
| ______________________________________ |
| P-23924A CH3 H OCH3 |
| P-23924B CH3 OCH3 OCH3 |
| P-23924C H OCH3 OCH3 |
| P-23924D CH3 OCH3 OH |
| P-23924E CH2 OH |
| H OCH3 |
| P-23924F CH2 OH |
| OCH3 OCH3 |
| ______________________________________ |
Having a free carboxyl group, compound fo of methanol was dissolved 1 g of P-23924A, and the solution is subjected to catalytic reduction with 5 g of Raney nickel under the initial pressure of 100 Kg/cm2 and at reaction temperature 70°C for 5 hours. Thus obtained reaction mixture was subjected to filtration to remove the catalyst, and the filtrate was concentrated to 20 ml under reduced pressure. The concentrate was diluted with water to give a 300 ml solution. The solution was extracted three times with 100 ml each portion of ethyl acetate, the extracts were combined and washed twice with 50 ml of water, the ethyl acetate layers were separated, dried and concentrated under reduced prressure to give 119 mg of orange red crystals of P-23924AR.
In 500 ml of methanol was dissolved 2.5 g of P-23924B, and the solution is subjected to catalytic reduction with 15 g of Raney nickel under the initial pressure of 100 Kg/cm2 and at the reaction temperature 70°C for 5 hours. Thus obtained reaction mixture was subjected to filtration to remove the catalyst, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in 250 ml of ethyl acetate. The solution was washed three times with 100 ml each portion of water, the ethyl acetate layers were separated, dried and concentrated under reduced pressure to give whereby 0.9 g of orange red crystals of P-23924BR.
In 700 ml of methanol was dissolved 3 g of P-23924C, and the solution is subjected to catalytic reduction with 15 g of Raney nickel under the initial pressure of 100 Kg/cm2 and at reaction temperature 70° C. for 5 hours. Thus obtained reaction mixture was subjected to filtration to remove the catalyst, and the filtrate was concentrated under reduced pressure to dryness. The residue was dissolved in 800 ml of ethyl acetate. The solution was washed three times with 300 ml each portion of water, the ethyl acetate layers were separated, dried and concentrated under reduced pressure to give whereby 1.23 g of orange red crystals of P-23924CR.
One gram of P-23924 D was treated with the manner of Example 3, whereupon 341 mg of orange red cyrstals of P-23924 DR was obtained.
| ______________________________________ |
| Tablet |
| ______________________________________ |
| (1) P-23924A, B, E or F |
| 100 mg |
| (2) Lactose 47 mg |
| (3) Corn starch 40 mg |
| (4) Hydroxypropylcellulose-L |
| 12 mg |
| (5) Magnesium stearate 1 mg |
| 200 mg/tablet |
| ______________________________________ |
A mixture of the above ingredients is tableted by the conventional method (wet method).
| ______________________________________ |
| Capsule |
| ______________________________________ |
| (1) P-23924C 100 mg |
| (2) Lactose 135 mg |
| (3) Corn starch 60 mg |
| (4) Magnesium stearate |
| 5 mg |
| 300 mg/capsule |
| ______________________________________ |
The above ingredients (1), (2), (3) and (4) (half the above amount) are mixed and granulated by the conventional method. To the granules is added the remaining amount of the ingredient (4). The mixture is packed into a gelatin capsule No. 1 (according to the Pharmacopoeia of Japan, Tenth Edition).
| ______________________________________ |
| Capsule |
| ______________________________________ |
| (1) P-23924D 300 mg |
| (2) Lactose 135 mg |
| (3) Corn starch 60 mg |
| (4) Magnesium stearate |
| 5 mg |
| 500 mg/capsule |
| ______________________________________ |
The above ingredients (1), (2), (3) and (4) (half the above amount) are mixed and granulated by the conventional method. To the granules is added the remaining amount of the ingredient (4). The mixture is packed into a gelatin capsule No. 00 (according to the Pharmacopoeia of Japan, Tenth Edition).
| ______________________________________ |
| Tablet |
| ______________________________________ |
| (1) P-23924 AR, BR, CR or DR |
| 100 mg |
| (2) Lactose 47 mg |
| (3) Corn starch 40 mg |
| (4) Hydroxypropylcellulose-L |
| 12 mg |
| (5) Magnesium stearate 1 mg |
| 200 mg/tablet |
| ______________________________________ |
A mixture of the above ingredients is tableted by the conventional method (wet method).
Ishimaru, Takenori, Okazaki, Hisayoshi, Ohta, Kazuhiko
| Patent | Priority | Assignee | Title |
| 5744623, | Aug 01 1994 | PHARMA MAR, S A | Terpene-quinones with antitumour activity |
| Patent | Priority | Assignee | Title |
| FR2523127, | |||
| GB2132643, |
| Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
| Apr 22 1991 | Takeda Chemical Industries, Ltd. | (assignment on the face of the patent) | / |
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